The Escherichia coli glnB gene encodes a central component of nitrogen regulation known as PII. PII interacts with three different proteins. The product of glnD, a bifunctional uridylyltransferase (UT) and uridylyl-removing enzyme (UR) catalyzes the uridylylation of PII when nitrogen is scarce and catalyzes the deuridylylation of PII-UMP when nitrogen is plentiful. Both the uridylylated and unmodified forms of PII also interact with the adenylyltransferase enzyme (ATase) that covalently modifies glutamine synthetase in response to nitrogen availability; PII stimulates the adenylylation (inactivation) of glutamine synthetase by the ATase, and PII-UMP stimulates the de-adenylylation (activation) of glutamine synthetase by the ATase. Finally, the unmodified form of PII interacts with the bifunctional kinase/phosphatase protein NRII, product of glnL (ntrB), eliciting a phosphatase activity that results in the dephosphorylation of the transcriptional activator NRI-Phosphate by the NRII/Pll phosphatase. This event in turn causes transcription at sigma 54-dependant nitrogen regulated promoters to cease. We propose to study the protein-protein interactions of the PII protein and its receptor proteins using genetic and biochemical methods. We describe a plan for the isolation and rapid sequencing of many glnB mutations that specifically destroy either the interaction with NRII or the interaction with the UT/UR. In addition, we describe genetic and biochemical methods designed to probe the PII-NRII interaction. The results of our study should help to elucidate the nature of the interactions between PII and the three other proteins that it contacts.